The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para-azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates “click” reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%–60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology.

翻译过程中非标准氨基酸 (nsAAs) 的特定位点掺入扩展了蛋白质的化学和功能。nsAA对叠氮基苯丙氨酸 (pAzF) 编码一个双正交化学部分，该部分促进“点击”反应以连接不同的化学基团以实现蛋白质功能化。然而，叠氮化物部分在生理条件下不稳定并被还原为对位-氨基苯丙氨酸 (pAF)。叠氮化物还原将蛋白质中 pAzF 残基的产量降低到每个叠氮化物的 50%–60%，并通过点击反应限制蛋白质功能化。在这里，我们描述了 pH 可调的重氮转移反应的使用，该反应将 pAF 转化为 pAzF，在蛋白质中的效率 >95%。该方法在每个蛋白质的多个位点选择性地恢复 pAzF，而不会引入脱靶修饰。这项工作解决了生产含 pAzF 蛋白质的关键限制，方法是恢复叠氮化物以实现具有不同化学部分的多位点功能化，为生产在生物治疗学、材料科学和生物技术中广泛应用的基因编码生物材料奠定了基础。